GSM5730131: CB-derived HSPCs - ASXL1 Trunc - H2AK119Ub - Rep1; Homo sapiens; OTHER
100,000 cells were washed twice in CUT&RUN wash buffer (20 mM HEPES, 150 mM NaCl, 0.5 mM Spermidine, supplemented with Roche EDTA-free cOmplete Protease Inhibitor). Next, activated Concanavalin A-coated magnetic beads (BioMagPlus Concanavalin A, Bang Laboratories, IN, USA) were added to the cell suspension and cells were allowed to bind to the beads for 10 min at room temperature under gentle agitation. After washing, the cells/bead mixture was resuspended in antibody buffer (consisting of wash buffer supplemented with 0.1% digitonin, 2 mM EDTA and 0.5 % BSA) and H2AK119Ub antibody (Clone D27C4) or IgG control (Clone DA1E, both Cell Signaling Technologies, MA, USA) at a 1:50 dilution and incubated with gentle agitation at 4°C overnight. After washing twice to remove unbound antibody, Protein A/G-MNase fusion protein (Epicypher, NC, USA) was added to the cell/bead mixture and allowed to bind for 1h at 4°C. Cells were washed twice to remove unbound antibody, resuspended in wash buffer supplemented with 0.1% digitonin, allowed to cool to 0° C in a chilled ice block before adding CaCl2 to a final concentration of 2 mM to initiate cleavage. After 30 min, the reaction was quenched with EDTA and EGTA and cleaved fragments were allowed to be released into the supernatant by incubating the cells at 37°C for 30 min. Fragments were cleaned up using Monarch PCR & DNA Cleanup Kit (NEB, MA, USA), sequencing libraries were prepared using the Ovation Ultralow V2 DNA-Seq Library Preparation Kit (Tecan, Männedorf, Switzerland) according to the manufacturer's protocol.